The genetic information of living organisms is carried in the nucleotide sequence of their genome. In the process of gene expression the nucleotide sequence is translated to amino acid sequences, i.e. proteins. Minor changes in the nucleotide sequence, even a single base substitution, may result in an altered protein product. The altered quality or quantity of given proteins changes the phenotype (i.e. the observable characteristics) of the organism or the cell, which for instance may be observed as a development of a disease.
The knowledge of the exact molecular defects causing inherited diseases, as well as predisposition to genetic disorders and cancer is increasing rapidly. The knowledge of the relevance of somatic mutations in malignancies is, however, limited due to the lack of rapid and reliable assay procedures for screening large numbers of samples.
Inherited diseases caused by point mutations include sickle cell anemia and β-thalassemias, which are caused by mutations in the β-globin gene. Antonarakis, 1989, New England J. Med., 320:153–163. These mutations generally involve the replacement, insertion or deletion of one to four nucleotides from the sequence of the normal gene. A large number of mutations in the β-globin gene that can lead to β-thalassemia have been characterized. Antonaraki, supra.
Other known inherited diseases caused by point mutations include α-thalassemia, phenylketonuria, hemophilia, α1-anti trypsin deficiency (Antonarakis, supra) and cystic fibrosis. Kere et al., 1989, Science, 245:1073–1080. Sickle cell anemia is caused by homozygosity for one unique base pair substitution in the sixth codon of the β-globin gene. Antonarakis, supra.
Cystic fibrosis is the most common autosomal recessive genetic disorder. It affects about 1/2000 individuals of Caucasian populations and consequently the carrier frequency is about 5%. The recent cloning and genetic analysis of the cystic fibrosis transmembrane regulator (CFTR) gene has revealed one major mutation, denoted ΔF508, which is a deletion of three nucleotides leading to loss of the phenylalanine at amino acid residue 508. Kerem et al., 1989, Science, 245:1073–1080. The prevalence of this mutation is on the average 68% in North American and European patient populations, the range being 40–88% in reports containing more than 100 CF chromosomes. Because of the high frequency of cystic fibrosis, efficient methods for the screening of carriers and for prenatal diagnosis are needed in the risk of group countries.
An example of a polymorphism which correlates to predisposition to disease is the three-allelic polymorphism of the apolipoprotein E gene. This polymorphism is due to single base substitutions at two DNA loci on the Apo E gene. Mahley, 1988, Science, 240:622–630. The polymorphism may explain as much as 10% of the individual variations in serum cholesterol levels. More than 90% of patients with type III hyperlipoproteinemia are homozygous for one of the Apo E alleles.
The human major histocompatibilty complex is a polymorphic system of linked genes located within a conserved region of the genome. The class II genes within the HLA-D (human leukocyte antigen) region encode a series of highly polymorphic alleles. Thomson, 1988, Annu. Rev. Genet., 22:31–50; Morel et al., 1988, Proc. Natl. Acad. Sci. USA, 85:8111–8115; and Scharf et al., 1988, Proc. Natl. Acad. Sci. USA, 85:3504–3508. This polymorphism has been shown to be associated with susceptibility to autoimmune diseases, such as insulin-dependent diabetes and pemphigus vulgaris.
The human ras-gene family, which includes the homologous H-, K- and N-ras genes, is one of the potential targets for mutational changes that play a role in human tumorigenesis. Point mutations in either codon 12, 13 or 61 of the ras genes have been shown to convert these genes into transforming oncogenes. Farr et al., 1988, Proc. Natl. Acad. Sci. USA, 85:1629–1633; and Bos et al., 1987, Nature, 327:293–297.
Somatic point mutations in the N-ras gene have been detected in association with acute myeloid leukemias (AML) and other hemotological malignancies). The N-ras mutations in AML occur predominantly in codons 12, 13 and 61 of the gene. A method for sensitive detection of the N-ras mutations in small quantities of leukemic cells amongst a vast majority of normal cells would constitute a most valuable tool in the follow-up of therapy of AML and other N-ras associated malignancies.
The detection of the specific base changes in the first and second position of codons 12, 13 and 61 of the N-ras gene requires either hybridization with a large number of different oligonucleotide probes, or direct nucleotide sequence determination of the amplified DNA. One critical aspect of both approaches is the proportion of cells containing the mutation. Depending on the method of choice a mutation must be present in 5–20% of the analyzed cell population to be detectable.
Point mutations and genetic variations in microorganisms might lead to altered pathogenicity of resistancy to the therapeutics. The human immunodeficiency virus (HIV-1) can develop mutants which are resistant to zidovudine (AZT). The resistant virus isolates contain several point mutations, but three mutations seem to be common to all resistant strains: Asp 67-Asn (GAC-AAC), Lys 70-Arg (AAT-GAT) and Thr 215-Phe (ACC-TTC) or Tyr (ACC-TAC). Larder and Kemp, 1989, Science, 246:1155–1158.
It would therefore be significant if changes in nucleotide sequences in the genome of living organisms could be determined accurately and with such efficiency and ease that large numbers of samples could be screened. This would afford opportunities for pre- or postnatal diagnosis of hereditary predispositions or diseases and for detection of somatic mutations in cancer. Such a method could also be used for the selection of cells and strains for industrial biotechnology and for plant and animal breeding. Presently available methods suffer from drawbacks limiting their routine use.
Polymorphisms or mutations in DNA sequences are most commonly detected by hybridization to allele-specific oligonucleotide (ASO) probes. The nucleotide sequence of the ASO probes is designed to form either a perfectly matched hybrid or to contain a mismatched base pair at the site of the variable nucleotide residues. The distinction between a matched and a mismatched hybrid is based on i) differences in the thermal stability of the hybrids in the conditions used during hybridization or washing (European Patent Publication EP-237362), ii) differences in the stability of the hybrids analyzed by denaturing gradient electrophoresis or iii) chemical cleavage at the site of the mismatch (European Patent Publication EP-329311).
Oligonucleotides with 3′ ends complementary to the site of the variable nucleotides have been used as allele-specific primers (European Patent Publication EP-332435). The identification of the variable nucleotide is based on the fact that a mismatch at the 3′ end inhibits the polymerization reaction. A similar approach is used in oligomer ligation assays, in which two adjacent oligonucleotides are ligated only if there is a perfect match at the termini of the oligonucleotides (European Patent Publication EP-336731).
Cleavage of the DNA sequence with restriction enzymes can be utilized for identification of the variation, provided that the variable nucleotide alters, e.g. creates or destroys, a specific restriction site. Nucleotide sequencing is the most informative method for the determination of variable nucleotides.
The methods referred to above are relatively complex procedures, suffering from drawbacks making them difficult to use in routine diagnostics. The use of allele specific oligonucleotide probes requires careful optimization of the reaction conditions separately for each application. Fractionation by gel electrophoresis is required in several of the methods above. Such methods are not easily automatized.